Study Of HBV DNA Integration In Oocyte/embryo And The ART Outcomes Of HBV Infected Women.

Background:Chronic hepatitis B is a major health problem caused by Hepatitis B virus (HBV) and about 2 billion people are exposed to HBV worldwide. Approximately 80% of primary liver cell carcinoma has relevance with HBV infection. China is recognized as one of HBV-endemic areas, and the prevalence of HBsAg is 7.2% in the population aged 1-60 years but approximately 10% in the group of childbearing age.HBV had been proved to present in some extra-hepatic tissues such as saliva, tears, sweat, and milk, semen, inner secretions and blood. Hepatitis B e antigen (HBeAg), HBV-DNA and Pre-S1 have been used clinically as an index of viral replication and infectivity. HBV DNA level is considered to be the most accurate marker for HBV replication. The transmission routes includs HBV through blood transfusion, body fluids contact and vertical transmission (father-child, mother-child). Mother-to-child transmission includes prenatal transmission, intrapartum propagation and postpartum spread. The last rout happens via generative cells or transplacental transmission. At present, it has been proved that most intrauterine infections could be prevented by HBV vaccine inoculation and hepatitis B immunoglobulin (HBIG) injection in the perinatal stage. However, about 10% of the infections cannot be prevented. It has also been confirmed that HBV DNA could integrated into the human chromosomes not only in hepatocellular carcinoma cells but also in spermatozoon, by which HBV was supposed to be transmitted to the embryos or fetus.HBV infection has been found to be associated with a reduced fertility. The HBV DNA integrating in sperm chromosomes might alter genetic constituents or induce chromosome aberrations and eventually cause hereditary effects. Systemic immunology or local immunological factors in generative organs caused by HBV infection may afffect human reproduction performance. Assisted reproduction techniques (ART) are increasingly being used for a variety of infertility problems. Assisted reproduction in infertile couples with HBV positive gives rise to many concerns about cross-contamination in laboratory and the ART outcomes of the HBV infected patients. However, there is no consensus on this issue.Our objective was to examine the HBV DNA in follicular and the relationship between HBV replication and HBV DNA integration in oocyte/embryo and the ART outcomes of HBV infected women.Materials and methods:Serum and follicular fluid from 176 HBsAg seropositive women who underwent IVF/ICSI-ET in our unit from 2009.02 to 2009.12 were obtained.206 unfertilized oocytes and 441 discarded embryos from 106 of the 176 women were collected. Fluorescence in situ hybridization (FISH) was carried out with biotin-labeled full length HBV DNA probe to confirm the integration of HBV in oocytes and embryos. Real time PCR was applied for HBV DNA in serum/in follicular fluid quantitative detection. The relationship between HBV replication and HBV DNA integration in oocyte/embryo were carried out using statistical software. A retrospective review on the IVF/ICSI and embryo transfer outcomes of 1114 cycles from 2004.01 to 2009.12 in our unit was carried out. A total of 557 cycles with the wives being seropositive but husbands seronegative for HBsAg were categorized as HBV (study) groups. The controls were matched with regard to the date of ova retrieval (day±1), female age (age±1), ART approaches (IVF or ICSI) and randomized by statistics software SPSS in the ratio of 1:1. Implantation rates (number of gestational sacs per embryo transferred), clinical pregnancy rates (presence of a gestational sac intrauterine per cycle with embryo transfer), miscarriage rates (number of miscarriages per clinical pregnancy) and delivering rates (number of childbearing women per cycle with embryo transfer) were analyzed. The relationship between HBV replication and the ART outcomes were also analyzed.Results:The serous HBV DNA positive rate was 54.4% and 45.7% of the follicular fluid samples were found to be HBV DNA positive. Generally, HBV DNA loads was higher in serum than in follicular fluid and serum Log10 HBV DNA showed moderate correlation with Log10 HBV DNA in FF; 8.25% of oocytes and 12.92% of embryos showed the signal of HBV DNA integration. Women with HBeAg seropositive or with serous HBV DNA≥106cop/ml or HBV DNA≥105cop/ml in follicular fluid showed higher rate of HBV DNA integration in oocyte and embryo; The HBV positive couples showed significantly lower delivering rates, and those patients with HBV DNA>105cop/ml in the serum had significantly higher miscarriage rate and lower live birth rate. Couples with HBV DNA in the follicular fluid showed lower implantation rate, lower clinical pregnancy rate, lower delivering rate and rate of taking baby home.Conclusions:1, HBV exists and duplicates in human follicular fluid;2, HBV DNA integrates in human oocyte and embryo;3, HBV DNA in follicular fluid is generally lower than that in serum and there is a moderate correlation of virus loads in serum and follicular fluid;4, Women with serous HBeAg seropositive have more chances for HBV DNA integration in oocyte,5, Women with serous HBV DNA>1.0×106copies/ml or HBV DNA≥1.0×105copies/ml in follicular fluid have more chances for HBV DNA integration in oocyte and embryo;6, HBV infection has no effect on ovarian function;7, Women with HBsAg seropositive have lower delivery rate (live birth) than HBsAg seronegative patients after IVF/ICSI and embryo transfer;8, Couples with HBV DNA≥1.0×105copies/ml in serum of wives result in higher miscarriage rate after embryo transfer;9, Couples with HBV DNA positive in follicular fluid of wives result in lower implantation rate and clinical pregnancy rate after embryo transfer;...